We believe this site might serve you best:

United States

Select a Different Country and Language

Promega's Cookie Policy

Our website uses functional cookies that do not collect any personal information or track your browsing activity. When you select your country, you agree that we can place these functional cookies on your device.

Related Resources

Related Articles

Abstract

Promega’s MagneHis™ Protein Purification System provides a simple, rapid and reliable method for the purification of polyhistidine-tagged, expressed proteins. Paramagnetic precharged nickel particles (MagneHis™ Ni-Particles) are used to isolate His-tagged proteins directly from a crude cell lysate using either a manual or automated procedure. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry can be used for the identification of His-tagged proteins. We describe an efficient sample preparation method for purified His-tagged proteins resulting in samples that can be directly analyzed by MALDI-TOF mass spectrometry.

Robin Hurst, Gary Kobs and Tonny Johnson

Promega CorporationPublication Date: 2003

Introduction

In recent years, mass spectrometry (MS) has become an indispensable proteomics tool(1)(2)
. Because of their ease of purification and lack of interference with protein function, His-tagged proteins have been combined with mass spectrometry in studies of structure and function(3)
, protein-protein interactions(4)
, post-translational modifications(5)(6)(7)
, molecular weight determination(8)
and peptide mapping. Unfortunately, methods normally used for His-tagged protein purification result in the presence of imidazole, and high concentrations of salt and detergents, which interfere with mass spectrometric analysis by causing high backgrounds (Figure 1, Panel A). However, modified washing and elution conditions can decrease the high mass spectrometry backgrounds for His-tagged proteins, as shown in Figure 1, Panels B (– DNA) and C (+ DNA). In this article we describe a method developed for the elution of His-tagged proteins from MagneHis™ Particles that reduces the salt and eliminates imidazole, so that His-tagged proteins can be analyzed more precisely by mass spectrometry.

Figure 1. MALDI-TOF analysis of MagneHis™ purified His-tagged GFP.

A T7 S30-based bacterial cell-free system was used in this experiment. In vitro translated His-tagged GFP was purified using the procedure described in the article. Panel A: His-tagged GFP purified with 0.5M imidazole-containing buffer. Panel B: Control reaction without His-tagged GFP DNA. Panel C: His-tagged GFP DNA purified using the modified wash and elution steps.

MALDI-TOF Analysis of MagneHis™ Purified Proteins

A protocol for preparation of His-tagged proteins for MALDI-TOF analysis, is shown in Table 1. This method can be used for the purification and elution of His-tagged proteins from bacterial cells or cell-free bacterial expression systems. Steps 1–3 describe the purification of His-tagged proteins as directed in the MagneHis™ Protein Purification System Technical Manual, #TM060, while Steps 4–7 describe a modified wash and elution procotol that reduces salt and eliminates imidazole.

Results

A comparison of the standard protocol (TM060 and Table 1, Steps 1–3) and the modified protocol (Table 1, Steps 4–7) for purification/elution of His-tagged Green Fluorescent Protein (His-GFP) expressed in a T7 S30 cell-free expression system and analyzed by MALDI-TOF, is shown in Figure 1, Panels A and C. Panel A is the MALDI-TOF spectrum of the His-GFP purified/eluted with the standard protocol (containing imidazole and high salt concentrations), whereas Panel C shows the MALDI-TOF spectrum of the His-GFP purified/eluted with the modified protocol (Table 1, Steps 4–7). Background peaks are reduced when using the modified protocol in Steps 4–7 of Table 1. SDS-PAGE analysis is shown in Figure 2 (– DNA in Lane 1, + DNA in Lane 2) using the modified protocol for purification/elution. The data in Figure 2 show that using the modified protocol, MALDI-TOF analysis is possible for His-tagged proteins expressed in a bacterial cell-free expression system.

Figure 2. SDS-PAGE analysis of His-tagged GFP purified from a T7 S30 bacterial cell-free system using the modified protocol.

Lane 1: In vitro coupled transcription and translation of a sample without DNA. Lane 2: In vitro coupled transcription and translation with His-tagged GFP DNA.

Conclusions

The development of a sample preparation method for His-tagged proteins purified using MagneHis™ Protein Purification System can drastically reduce the background levels in MALDI-TOF mass spectrometry analysis. This method can be used for the analysis of His-tagged protein expressed in bacteria in vivo as well as in bacterial cell-free expression systems.

Products may be covered by pending or issued patents or may have certain limitations on use.

SpeedVac is a registered trademark of Savant Instruments, Inc.

Figures

Figure 1. MALDI-TOF analysis of MagneHis™ purified His-tagged GFP.

A T7 S30-based bacterial cell-free system was used in this experiment. In vitro translated His-tagged GFP was purified using the procedure described in the article. Panel A: His-tagged GFP purified with 0.5M imidazole-containing buffer. Panel B: Control reaction without His-tagged GFP DNA. Panel C: His-tagged GFP DNA purified using the modified wash and elution steps.

Figure 2. SDS-PAGE analysis of His-tagged GFP purified from a T7 S30 bacterial cell-free system using the modified protocol.

Lane 1: In vitro coupled transcription and translation of a sample without DNA. Lane 2: In vitro coupled transcription and translation with His-tagged GFP DNA.

Scientists at Your Service

We offer a range of services to help you succeed using Promega technologies. From product training to set up of automated systems and development of custom applications—our scientific support goes beyond the basics.